HOW IS GENETIC MATERIAL TRANSFER IN BACTERIA ....?

 What is bacteria ...?

 Single celled microorganisms that can exist either as independent organisms or as parasite. 

  eg.  E.coli , streptococcus ,bacillus ,lactobacillus .

What is mean by transfer of genetic material ...?

The insertion of unrelated genetic information in the form of DNA into cell. There are different reasons to do gene transfer.

There are three method of gene transfer in bacteria :-

1) Conjugation 

2) Transformation 

3) Transduction 

1) Conjugation :-

Conjugation is the transfer of a plasmid or other self-transmissible DNA element and sometimes chromosomal DNA from a donor cell to a recipient cell via direct contact usually mediated by a conjugation pilus or sex pilus.

Recipients of the DNA transferred by conjugation are called transconjugants.

The process of conjugation can transfer DNA regions of hundreds to thousands of kilobases and has the broadest host range for DNA transfer among the methods for bacterial exchange.

Step in conjugation :-

• The F plasmid contains tra locus, which includes the pilin This gene, along with some regulatory proteins results in the formation of pilli on the F⁺ cell surface.
• The proteins present in the pilli attach themselves on the F^– cell surface. The pilli are responsible for making contact between the cells, but the transfer of plasmid doesn’t occur through the pilli.
• The traD enzyme, located at the base of the pilus, initiates membrane fusion.
• Once the conjugation is initiated, enzyme relaxase creates a nick in the conjugative plasmid at the oriT
• The nicked strand (called the T strand) then unwinds and is transferred to the recipient cell in the 5’-3’ direction.
• The complementary strand is synthesized in both cells; thus, both the donor and recipient are F⁺.
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• In certain F⁺ bacterial cells, the F element infrequently (about once in every 10,000 F⁺ cells) becomes associated with the main bacterial chromosome in such a way that a copy of the chromosome instead is transferred through the conjugation tube from donor to recipient cell.
• In the insertion process, the circular F element breaks at a particular point and becomes a linear segment of the bacterial chromosome.
• An F⁺ cell that carries such an integrated F element is known as an Hfr cell (Hfr stands for the high frequency of recombination).
• The integrated F element of Hfr cells is ordinarily replicated passively along with the bacterial chromosome and in this way is transmitted from one Hfr generation to the next. 

2) Transformation :-

Bacterial transformation is the transfer of free DNA released from a donor bacterium into the extracellular environment that results in assimilation and usually an expression of the newly acquired trait in a recipient bacterium.

Step :-

There are four steps in transformation:

development of competence,

binding of DNA to the cell surface,

processing and uptake of free DNA (usually in a 3’ to 5’ direction), and

integration of the DNA into the chromosome by recombination.

The artificial development of competence can be achieved either through electroporation or through heat shock treatment. The choice depends on the transformation efficiency required, experimental goals, and available resources.

For heat shock, the cell-DNA mixture is kept on ice (0°C) and then exposed to 42°C.

For electroporation, the mixture is transferred to an electroporator and is exposed to a brief pulse of a high-voltage electric field.

The double-stranded DNA released from lysed cells binds noncovalently to cell surface receptors. There is no DNA sequence-specific recognition; thus, these organisms can potentially incorporate DNA from outside their species.

The bound double-stranded DNA is nicked and cleaved into smaller fragments by membrane-bound endonucleases, allowing the single strand to enter the cell through a membrane-spanning DNA translocation channel.

The transformed DNA integrates into the chromosome and replaces the chromosomal DNA fragment by recombination. This integration, however, requires significant nucleotide sequence homology between the donating DNA fragment and the fragment in the chromosome.

In the case of plasmid, the plasmid with the donor DNA is inserted during the heat shock or electroporation. The cells with the plasmid can be detected by growing these cells is a growth media supplemented with a specific antibiotic.

3) Transduction :-

Transduction, a process of genetic recombination in bacteria in which genes from a host cell (a bacterium) are incorporated into the genome of a bacterial virus (bacteriophage) and then carried to another host cell when the bacteriophage initiates another cycle of infection.

Types of Transduction

Based on how the DNA is packaged within the viral particle, there are two types of transduction:

 1) Generalized Transduction :- 

In generalized transduction, phage mistakenly packages bacterial DNA instead of their own phage DNA during phage assembly.

This results in an infectious virus particle containing bacterial DNA, but one that can no longer replicate in the bacterium due to the loss of all of the phage DNA.

The phage particle then attaches to a bacterial cell surface receptor and injects the packaged DNA into the cytoplasm of the bacterium.

If the bacterial DNA in the phage is from the bacterial chromosome, the DNA recombines with the homologous DNA of the bacterial recipient to generate stable transductants. This process requires a host recombinase, recA.

However, studies have indicated that the majority of transduced DNA is not stably integrated into the bacterial genome but rather remains extrachromosomal.

Generalized transduction is used for mapping genes, mutagenesis, transferring plasmids and transposons, and determining whether different genera of bacteria have homologous genes.

2) Specialized Transduction :- 

In specialized transduction, the phage undergoes lysogeny usually at specific locations in the bacterial genome called attachment sites.

During this process, the phage genome usually integrates into the bacterial chromosome as virus replication is repressed during lysogeny.

The phage genome then excises from the bacterial genome and, due to imprecise excision and recombination, adjacent bacterial genes are also excised.

Unlike a generalized transducing phage, a specialized transducing phage contains both phage and bacterial genes.

During the subsequent infection, the newly acquired gene is inserted into the bacterial genome along with phage DNA to form a new round of lysogeny.

Specialized transduction is independent of host homologous recombination and recA but requires phage integrase.

Specialized transduction is instrumental in the isolation of the genes in molecular biology, and in the discovery of insertion elements, which often serve as attachment sites for phage DNA integration.

Steps of Generalized Transduction :- 

The phage host cell (donor cell) is first infected with the phage, during which, the phage DNA enters the cytoplasm of the bacteria.

During the lytic cycle of the viral replication, the phage DNA, along with the bacterial chromosome is broken down into smaller pieces.

Some part of the bacterial chromosome is then packaged into one of the viral capsids when that is released by lysis of the bacterium.

The transducing phase with the bacterial chromosome now infects a second bacterium (recipient bacterium), and the donor DNA enters the cytoplasm of the second bacterium.

In the presence of a host recombinase recA, the donor DNA recombines with the homologous DNA of the bacterial recipient to generate stable transductants. 

Steps of Specialized Transduction :- 

After the infection of the donor bacterium with the bacteriophage, the phage DNA is integrated into the bacterial chromosome during the lysogenic cycle.

Due to the imprecise cutting of the phage DNA, some part of the bacterial chromosome is also excised.

The phage containing some part of the bacterial chromosome then infects a new host, and the donor DNA is incorporated into the recipient bacterium during the lysogenic cycle of the replication.

The recipient then expresses the newly acquired genetic trait.